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Fig. 3 | Clinical Epigenetics

Fig. 3

From: MeCP2 and the enigmatic organization of brain chromatin. Implications for depression and cocaine addiction

Fig. 3

a After micrococcal nuclease (MNase) digestion of cellular nuclei, small-sized nucleosomes chromatin (nI) (see Fig. 4) that leak through the nuclear membrane pores can be recovered in the supernatant (SI) after centrifugation. The nuclear pellet can next be hypotonically lysed in the presence of 0.25 mM EDTA and centrifuged once more to yield a supernatant (SE) fraction and an insoluble pellet (P). b Protein composition of the SI, SE and P fractions as analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS detergent. Histones H1, H2A, H2B, H3 and H4 are indicated, as well as myelin (M). c Analysis of the SI, SE and P fractions during a time-course Mnase digestion of rat whole brain nuclei. The upper part of the Figure shows a Western blot analysis using MeCP2 and H4 antibodies. The lower part shows a native PAGE analysis of the DNA composition of the fractions obtained at different time of digestion. CE: chicken erythrocyte histones used as a control; M: pBR322-Cfo I–digested DNA used as a marker. The numbers on the right had side of the native PAGE indicate the DNA fragment sizes in base pairs (bp). The red lines highlight the shift in the center of the mononucleosome DNA (nI) distribution in SE and P. d Relative meC/C percentile composition of the SI, Se and P fractions at limit Mnase digestion. (Section c was reproduced from Fig. 2A from [9], with permission)

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